Antibodies (Abs) recognizing unique conformational epitopes only seen on functional HIV trimeric envelope are thought to be important for HIV neutralization and may be the key determinant of selection pressure on envelope (Env) viral sequences. We have created a panel of candidate Env trimer-specific Abs from single-cell sorting B cells from HIV positive subjects. I propose work to define important Ab structural determinants necessary for recognition of HIV trimeric Env and, in doing so, to provide insights for design of an effective HIV vaccine. I hypothesize that continued affinity maturation of Env trimer-specific Abs, rather than Env monomer- specific Abs, is the principal driving force for Env sequence diversity during HIV infection. I will study these novel native human Ab sequences by cloning them into Fab and full-length Ab expression systems. I will then use these constructs to define the biochemical and structural features of neutralizing Abs. In specific aim 1,1 will compare the binding and neutralization characteristics of Env monomer-reactive Abs to Env trimer-specific Abs. 1 will use surface plasmon resonance to compare binding and the infectible reporter TZM-bl cell line to assess neutralization. We expect to show the Env trimer-specific Abs bind and neutralize more effectively than Env monomer-reactive Abs. In specific aim 2,1 will determine the relationship of binding kinetics and neutralization potency to somatic hypermutation. It follows from my hypothesis and preliminary data that Abs possessing large numbers of mutations are likely being created by the process of affinity maturation, and hence, are improved in their binding and neutralization capabilities. Interestingly, most of the observed mutations occur outside of the major determinant of Abs binding, the CDR3 loop. Specific aim 3 is designed to define the role of somatic mutation outside of the CDR3 loop in HIV neutralization. Hybrids of mutagenized Abs with germline Ab sequences will be created by site-directed mutagenesis. I will compare binding and neutralization of the native Abs to these hybrid Abs. Because of the extent of mutation, we expect to show a critical role for regions outside of the CDR3 loop, particularly heavy chain framework 3, in HIV neutralization. RELEVANCE: The overall aim of this work is improvement in design of vaccines to produce higher potency antibodies against HIV. This work will directly study natural antiodies formed during HIV infection to find how they bind virus. Not only may this help improve our knowledge of how the immune system reacts to HIV, but it may shed light on how we can improve components of HIV vaccines and other vaccines to increase their potency.